GSM7385378: Kidney, fibroblasts, Trf1flox/flox; Col1a2-CreERT2, RGQ00355; Mus musculus; RNA-Seq
Mice were euthanized after 8 weeks and kidneys were extracted.  To isolate fibroblasts populations in Col1a2 -CreERT2 mice, kidneys were collected in PBS, minced, and digested with 2.5 mg/ml type I collagenase (Thermo Fisher, 17100017), 2.5 mg/ml dispase II (Thermo Fisher, 17105041), and 50 ng/ml DNAse I (Roche, 10104159001) at 37 °C for 20 min to create a homogeneous cell suspension. Cell suspensions were passed through a 70 μm filter (Cultek, 45352350) to remove any undigested tissue. To remove erythroid cells, cell suspensions and blood samples were incubated for 10 min on ice in blood erythrocyte lysis buffer (Qiagen, 79217). Before analysis, cell suspensions were treated with PBS 0.04% Ultrapure BSA (Thermo Fisher, AM216). Cells were incubated at 4 °C for 30 min with anti-mouse CD29-PE (clone: HMB1-1, 1:00) (Milteneyi Biotec, 130-102-994)  and anti t-RFP (EVN-AB233, Evrogen; 1:00) for fibroblasts isolation respectively. Chicken anti-Rabbit Alexa 647 (A21443, Invitrogen;1:400) was used as a secondary control against t-RFP Thermo Fisher, A21443. Isolated cells were collected on a mix of NEBNext® Cell Lysis Buffer (New England Biolabs, E6428A) and Murine RNase Inhibitor (New England Biolabs, E6429A) in DNA LoBIND tubes (Eppendorf, 0030108051), using a BD FACSAria IIu cell sorter (BD Biosciences). 500 cells per sample were were processed with the "NEBNext Single Cell/Low Input RNA Library Prep" kit (NEB #E6420) by following manufacturer instructions. Briefly, extracts obtained by sample suspension in cell lysis buffer were submitted to an oligo(dT) primed reverse transcription with a template switching reaction. Double stranded cDNA production was performed by limited-cycle PCR. Sequencing libraries were completed with the "NEBNext Ultra II FS DNA Library Prep Kit for Illumina" (NEB #E7805). These libraries are non-directional or non-stranded. The resulting purified cDNA libraries were applied to an Illumina flow cell for cluster generation and sequenced on an Illumina NextSeq 550 (with v2.5 reagent kits) by following manufacturer's protocols. Single read 86bp